Three dimensional growth of animal cell by new immobilization technique and its application for artificial liver

为了建立人工肝，人们尝试了肝细胞的长期传代。以胶原酶法分离的肝细胞为材料，研究了几种维生素和氨基酸对细胞维持的影响。其中，作为胶原主要成分的脯氨酸和激活胶原生物合成的辅助因子抗坏血酸，至少能促进白蛋白的生物合成一周。另一方面，分解有毒超氧阴离子的超氧化物歧化酶也促进了白蛋白的分泌。我们还研究了适合肝细胞固定化的材料，并开发了一种与肝素或胶原结合的大孔纤维作为细胞附着配体。Vero细胞与基质附着良好，细胞密度大于10^7/ml。这一结果表明该支架可能适用于人工肝的构建。为了构建人工血管，我们分离了人内皮细胞。用SV40病毒的温度敏感突变体感染细胞，在33ﾟC时，细胞寿命延长200天以上。这些细胞在37ﾟC时死亡，这是因为温度敏感的肿瘤蛋白在该温度下失活。由于这些细胞表达内皮细胞特有的功能，它们对血管相关疾病的分析是有用的。

In order to establish artificial liver, long term passage of liver cells has been tried. Using liver cells isolated by collegenase method, effects of several vitamins, and amino acids for cell maintenance were studied. Among them, proline which is main component of collagen, and activated ascorbate which is a cofactor of collagen biosynthesis, facilitated albumin biosynthesis at least for one week. On the other hand, super oxide dismutase which decompose toxic super oxide anion also facilitate albumin secretion.We also studied the suitable materials for immobilization of liver cells and developed a macroporous fiber which bound to heparin or collagen as cell attachment ligands. Vero cell attached very well to both of matrix and cell density was more than 10^7/ml support. This result indicated that the support may be suitable for artificial liver construction.For the construction of artificial blood vessel, we isolated human endothelial cells. By the infection of a temperature sensitive mutant of SV40 virus, life span of cells was elongated for more than 200days at 33ﾟC. These cells died at 37ﾟC since the temperature sensitive tumor protein was inactivated at this temperature. Since these cells expressed endothelial cell specific function, they are useful for the analyses of blood vessel related disorder.